PROJECT SUMMARY Our long-term goal is to understand how to remove macrophages and cholesterol from plaques to promote disease regression. We recently discovered that lymphatic vessels serve as the conduits by which cholesterol is removed from the artery wall and other tissues. Reverse cholesterol transport (RCT) is the process by which cholesterol is mobilized from the body for excretion through the feces. With respect to atherosclerosis, the mobilization of cholesterol from macrophages for removal and excretion is most relevant. Over the years, many details emerged regarding how cholesterol is mobilized from macrophages to be loaded onto HDL (HDL-C). However, little was known about how HDL-cholesterol subsequently makes its way out of tissues, including sites like atherosclerotic plaques of the artery wall, to return to plasma before entering pathways for excretion. A handful of clinical or experimental observations led a few physicians in the early 1980's to propose a connection between impaired lymphatic transport and atherosclerosis. However, the quantitative importance of lymphatic vessels in RCT had not been examined, aside from a compelling study that estimated that the net flux of HDL-C through human lymph is substantial. Recently, we utilized experimental mouse models where the patency of lymphatic flow could be modulated by surgical or genetic methods. In skin, we were able to fully abrogate lymphatic flow allowing us to demonstrate that lymphatics are quantitatively the major route for cholesterol mobilization to plasma following macrophage RCT. In atherosclerosis-affected aortic walls, we also used a surgical approach to track deuterium-labeled cholesterol ([2H] D6-cholesterol; D6-cholesterol) from plaques in a pulse-chase manner. Aortas were surgically transplanted into recipients with re-anastomosis of the lymphatic vasculature blocked or not with anti-VEGFR3 mAb. This blockade significantly retained D6- cholesterol in the atherosclerotic aorta, suggesting a key role for lymphatic vessels in cholesterol mobilization from the aorta as observed in skin. In aim 1, we will take a critical next step with refined approaches that will allow us to better quantify the role of lymphatic vessels in cholesterol removal from the aorta and to assess whether the blockade on lymphatics is truly acting locally at the aortic wall. In preliminary data, we show that treating apoE-/- mice with VEGF-C, the ligand for VEGFR3, restores impaired lymphatic transport that occurs following hypercholesterolemia, allowing us to test the hypothesis that VEGF-C acts on lymphatic vessels to therapeutically sustain a critical route for cholesterol transport out of plaques after macrophage cholesterol efflux is stimulated. This hypothesis raises a fundamental question not yet addressed in the field: does effective plaque regression truly depend upon cholesterol removal from plaques, only upon cholesterol removal from macrophages, or perhaps neither? Our study design is ideal to address this fundamental issue while simultaneously digging deeply into a new concept that supporting lymphatic vessel function may help resolve inflammation in the atherosclerotic plaque.